Alpha/1-Adrenergic receptors (alpha/1-AR) mediate important effects f the sympathetic nervous system, such as arteriolar constriction. Three alpha/1-ARs have been cloned. However, the physiological significance of this subtype diversity remains unclear, since they are functionally similar in their ligand recognition and signaling properties, and are often coexpressed. The goal of this proposal is to more clearly define their physiological function and investigate possible differences between these subtypes. In our efforts to understand signal transduction by alpha/1-ARs, we intend to focus on a number of mutants that can clarify which residues are involved in subtype selectivity, and if a particular residue(s) is involved in transducing a signal to the G-protein. Secondly, studies will be performed by developing a model system to study alpha/1-AR subtype inactivation by gene targeting. The FRTL-5 cell line is an ideal model system since its alpha/1-AR signaling characteristics and receptor-mediation of thyroid metabolism have been well characterized. In this cell line only a single alpha/1-AR (alpha/1B) appears to be expressed that can couple to its effector pathways via two distinct G- proteins. However, expression of another subtype(s), at low level, cannot be excluded. To test this hypothesis, I propose to inactivate the alpha/1B-AR and then assess its role in thyroid regulation. The generation of an alpha/1B-AR deficient cell line will also be advantageous in the analysis of the alpha/1B-AR mutants generated in Specific Aim 1. Specifically, our aims are the following: i) To undertake structure-function studies using site-directed mutagenesis in determining which residues are involved in subtype selectivity. ii) To fully characterize and understand the properties of a constitutively activated alpha/1B-AR that has been mutated in the third transmembrane domain. iii) To achieve a double allele disruption of the alpha/1B-AR gene by homologous recombination in somatic FRTL-5 cells and generate an alpha/1B- AR deficient cell line for mutational studies.